We published four papers related to this project in the last cycle. One finding was: "Based on non-competitive binding interactions we suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. We used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. (3)Hd-Ala(2),d-Leu(5)enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased (3)Hd-Ala(2),d-Leu(5)enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease (3)Hd-Ala(2),d-Leu(5)enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: d-Pen(2),d-Pen(5)enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that d-Pen(2),d-Pen(5)enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both d-Pen(2),d-Pen(5)enkephalin and morphine competitively inhibited (3)Hd-Ala(2),d-Leu(5)enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers."